Nanopore-facilitated single molecule detection of nucleic acid

ABSTRACT

The present invention provides a new and improved oligonucleotide detection method based on the nanopore technology with a probe containing a complementary sequence to the target oligonucleotide and a terminal extension at the probe&#39;s 3′ terminus, 5′ terminus, or both termini. The improved nanopore sensor with the probe enables sensitive, selective, and direct detection, differentiation and quantification of target oligonucleotides such as miRNAs. The inventive detection method may also be employed as a non-invasive and cost-effective diagnostic method for cancer detection based on miRNA levels in the patient&#39;s blood sample.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationNo. 61/399,578, filed Jul. 14, 2010, which is incorporated herein byreference in its entirety.

GRANT STATEMENT

This invention was made with government support under Grant No. GM079613awarded by the National Institute for Health. The government has certainrights in the invention.

FIELD OF INVENTION

This product relates to a method/apparatus of single-molecule detection,more specifically, to a method/system for quantitative detection ofsingle strand nucleic acids, such as microRNAs, employing anultrasensitive, low noise nanopore-based single-molecule technology.

SEQUENCE LISTING STATEMENT

The sequence listing that is contained in the file named“52553_(—)96854_ST25.txt”, which is 12303 bytes in size (measured inoperating system MS-Windows), created on Jul. 14, 2011, is filedherewith by electronic submission and incorporated herein by referencein its entirety.

BACKGROUND OF INVENTION

MicroRNAs.

MicroRNAs (miRNAs) are a class of short (˜18-24 nucleotides) noncodingRNAs that regulate gene expression at the post-transcriptional level².Depending on the degree of homology to their target sequences, miRNAbinding induces either translational repression or cleavage of targetmRNAs². As powerful gene regulators, miRNAs play important roles indevelopment, cell differentiation, and regulation of cell cycle,apoptosis and signaling pathways^(2,3). Aberrant expression of miRNAshas been found in all types of tumors^(4,5); the different cancer typeshave distinct miRNA expression profiles⁶. Specific miRNAs including somemiRNA families containing a few nucleotide differences are constantlyreleased from the primary tumor into blood stream and present in anincredible stable form⁷. Recent studies demonstrated that circulatingmiRNAs are enveloped inside exosomal vesicles and can be transferableand functional in the recipient cells^(8,9). Thus, detection of tumorspecific circulating miRNAs provides a powerful tool for earlydiagnosis, staging, and monitoring of cancer cells¹⁰.

MiRNA Detection.

Several technologies including reverse transcription real-time PCR(RT-qPCR) and microarray for miRNA detection have been developed¹¹⁻¹³.Each technology has its own advantages, but limitations includerequiring enzymatic amplification and semi-quantitative results¹⁴. Inparticular the short miRNA sequences make it difficult to selectivelydesign the primers or probes, resulting in cross-hybridization and lowselectivity. This is especially true when the miRNAs contain a few or asingle nucleotide difference in a miRNA family. Emerging techniquesbased on colorimetry, bioluminescence, enzyme turnovers andelectrochemistry have been proposed, and nanoparticles and molecularbeacon have been applied to miRNA detection with high sensitivity andselectivity (review¹⁴). But the intrinsic versatility needs to beimproved. Recently, the integration of single-molecule fluorescence andlock-nucleic acids (LNA)¹⁵ probes provided a sensitive method for miRNAprofiling in tissue samples¹⁶, though this method requires expensiveinstrument.

Nanopore Single Molecule Detection.

In a nanometer-scaled pore structure, the ion current becomes verysensitive to the presence, location and conformation of single targetmolecules occupying the ion pathway¹⁷. This sensitivity allows“visualizing” single molecules, elucidating their kinetics fromcharacteristic change in the pore conductance, and quantifying thetarget from the occurrence of single molecule signature events.Nanopores have been developed as receptive single molecule detectors forbroad biotechnological applications (reviews¹⁷⁻¹⁹). The nanopore is alsorecognized as one of the next generations of DNA sequencingtechnologies^(20,21). For example, the 2-nm nanopore, α-hemolysintransmembrane protein pore, allows rapid translocation ofsingle-stranded oligonucleotide, which has been well characterized forDNA sequencmg²²⁻²⁷. However, the molecular translocation-based sensingmode is not suitable for miRNA detection because the sequences of allmature miRNAs are short (18-24 nt), and when traversing the nanopore,the current signals by different miRNAs are indistinguishable.

Therefore, there is a need to provide a new miRNA detection method basedon nano-scale pore structure with improved sensitivity, speedy process,and cost efficiency.

SUMMARY OF INVENTION

In one aspect of the invention, a new and improved nanopore-basedsensing system for detection and differentiation of single strandoligonucleotides, such as miRNAs, is described. The inventive system fordetecting a target single strand oligonucleotide comprises 1) ananopore, 2) a power source providing sufficient voltage to induceunzipping, 3) a probe with its center domain complementary to the targetoligonucleotide, whereas the unzipping of the hybrid of targetoligonucleotide and the probe in the nanopore induces certainidentifiable current signal changes, and 4) means for detecting thecurrent signal changes. The inventive probe further comprises at leastone signal tag at its 3′ or 5′ terminal (or both). The signal tag may beof any charged single chain molecule with sufficient length to assistthe unzipping translocation through the nanopore driven by the voltage.For example, the signal tag may be oligonucleotides such aspoly(dC)_(n), poly(dA)_(n), and poly(dT)_(n), or charged polypeptides.

In another aspect of the invention, a new and improved method based onnanopore technology for detecting and differentiating single strandoligonucleotide is described. The inventive method detects the currentchanges triggered by the unzipping of the hybrid of the targetoligonucleotide and its probe in a nanopore. The inventive methodincludes the step of 1) mixing the target oligonucleotide with apre-designed probe, which has its central domain matching the targetsequence and a charged single chain molecule tagged to at least one ofits 3′ and 5′ terminals, to produce a sample mixture, 2) loading themixture into the cis chamber of a nanopore system, and a voltage isadded from the trans chamber, and 3) recording current output for apre-determined time period.

In yet another aspect of the invention, a new and improved method fordetecting and monitoring cancer-related miRNAs in patients' blood sampleis described. The inventive method includes the steps of 1) mixing thetotal plasma RNAs extracted from a patient's blood sample with the miRNAprobe that contains the complementary sequence to the targeting miRNAand a signal tag at the probe's 3 ‘-terminal, 5 ’-terminal, or both, 2)adding the mixture into a nanopore chamber with a preselected voltage,and 3) monitoring and analyzing the signature events in the outputcurrent traces that serves as an electrical marker for single miRNAmolecule recognition.

In certain embodiments, a probe molecule for detecting of a singlestrand oligonucleotide, such as miRNA, using a nanopore comprising: 1) acenter domain with a complementary sequence to the targetoligonucleotide, and 2) a terminal extension tagged to at least one ofthe center domain's 3′ or 5′ terminals is provided. In certainembodiments, the terminal extension is a charged chain molecule. Incertain embodiments, the terminal extension is a charged polypeptide. Incertain embodiments, terminal extension is a charged polymer.

In certain embodiments, the invention provides a method of detectingsingle strand oligonucleotide with a dual-compartment nanopore system,whereas the system includes a cis compartment and a trans compartmentdivided by a partition with an opening at its center region, recordingsolution filling both compartments and a lipid bilayer formed at theopening on the partition, a nanopore plugging through the lipid bilayerbridging the cis and trans chamber, a voltage loaded upon the system viaa pair of electrodes each extruding from the cis or trans compartment,and a current detector monitoring the current changes, includes thesteps of 1) mixing the target oligonucleotide with a pre-designed probe,which has its central domain matching the target sequence and a chargedsingle chain molecule tagged to at least one of its 3′ and 5′ terminals,to produce a sample mixture, 2) loading the mixture into the ciscompartment, 3) providing the system with a pre-determined voltage, and4) recording current output for a pre-determined time period. In certainembodiments of the methods, the recording step can further comprise thestep of analyzing the current change induced by the hybrid of the targetoligonucleotide and the probe undergoes unzipping in the nanopore.

The instant invention also includes probes, nanopores, kits comprisingthe probes and nanopores, and associated methods of use described in thefollowing portions of the specification, drawings, and claims providedherewith.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of an exemplary nanopore sensing system,according to one embodiment of the invention.

FIG. 2 is a schematic amplified illustration of an exemplary nanoporeplugged through the lipid bilayer.

FIG. 3 includes an exemplary current trace, an amplified electrical markof the signature event, and a schematic illustration of the unzippingand translocation event.

FIGS. 4(A) to (D) illustrate an exemplary detection of miR-155 with theinventive probe, P₁₅₅: FIG. 4(A) is a current trace showing differenttypes of blocks. The block profiles and corresponding molecularprocesses is depicted in panel B, C and D; FIG. 4( b)/(b′) is anexemplary spike-like short block produced by free miR-155 or P₁₅₅molecules translocating through the pore; FIG. 4( c)/(c′) is anamplified long block with multiple conductance levels which aresequentially generated by unzipping of a miR-155·P₁₅₅ hybrid,confinement of miR-155 in the nanocavity and translocation of miR-155;FIG. 4( d)/(d′) is an exemplary long blocks with a single conductancelevel, produced by a trapped mir-155·P₁₅₅ hybrid that exits from the cisentrance without unzipping.

FIGS. 5( a) and (b) illustrate the invention employed in thequantification of miRNAs using the nanopore sensor. a. Current traces inthe presence of 100 nM P₁₅₅ and different concentrations of miR-155. Thesignature events for miR-155·P₁₅₅ hybrid interacting with the pore aremarked with red arrows. b. Correlation between the miR-155 concentration[miR-155] and the frequency of signature events (f_(sig)). Significance(p<0.01) is valid between detections in any two miR-155 concentrations.The f_(sig)-[miR-155] curve is fitted using Eq. 1.

FIGS. 6( a) and (b) illustrate the invention employed in thedifferentiation of miRNAs with similar sequences. a. Current traces fordetections of let-7a and let-7b using the probe P_(a) (+120 mV). b.Durations of signature events (τ_(sig)) for let-7a·P_(a), let-7b·P_(a),let-7a·P_(b) and let-7b·P_(b).

FIGS. 7( a) to (f) illustrate the invention employed in the detection ofmiR-155 in lung cancer patients' plasma. a through d, the signatureevents were found in current traces for total plasma RNAs from normalvolunteers (a) and lung cancer patients (b) in the presence of 100 nMP₁₅₅ probe, but not observed in the absence of P₁₅₅ (c and d). Thetraces were recorded in 1 M KCl at +100 mV. e. Frequencies of miR-155signature events (f_(sig)) from six normal individuals (N1 to N6) andsix patients with lung cancer (P1 to P6). Each sample was measured ntimes (n≧4) with independent nanopores. The data was given as mean±SD.The patient conditions are, P1, metastatic squamous lung carcinoma; P2,recurrent small-cell cancer; P3, early stage of small-cell carcinoma,status post chemotherapy and radiation; P4, early stage of small-cellcancer, status post chemotherapy; P5, late stage non-small cellcarcinoma, statue post resection and chemotherapy; P6, late stageadenocarcinoma, status post chemotherapy. f. Relative miR-155 levels innormal group (left) and lung cancer patient group (right), measured withthe nanopore sensor and qRT-PCR. The means with SD were shown.

FIG. 8 A-G shows the capture of single miRNA molecules with a specificprobe in the nanopore. A. Molecular diagram of the miRNA·probe hybrid;B. Sequentially-occurred nanopore current blocks in the presence of 100nM miR-155/P₁₅₅ mixture in the cis solution. The recording solutioncontained 1 M KCl buffered with 10 mM Tris (pH 8.0). Traces wererecorded at +100 mV. The identified current patterns and correspondingmolecular mechanisms were depicted in panel c, f and g. The framedblocks demonstrated the multi-level current pattern depicted in panel cand d; c. Multi-level long block at +100 mV, generated by themiR-155·P₁₅₅ hybrid that was trapped in the pore, unzipped, followed bysequentially translocation of unzipped P₁₅₅ and miR-155 through thepore; d. Characteristic multi-level long blocks at +150 mV and +180 mV;e. qRT-PCR-detected miR-155 levels in cis and trans solutions after ˜6hours electrical recording at different concentrations of miR-155 andP₁₅₅ presented in the cis solution (Text in Supplementary Information);f. Single-level current pattern generated by a trapped mir-155·P₁₅₅hybrid that exited the pore from the cis entrance without unzipping; g.Spike-like short block generated by translocation of un-hybridizedmiR-155 or P₁₅₅ from the cis solution.

FIG. 9 A-B shows enhancing detection sensitivity by optimizing the probesequence. A. Left, current traces showing the frequency of signatureevents for miR-155 hybridized with the probes P_(5′-C30) (top),P_(3′-C30) (middle) and P₁₅₅ (bottom), monitored at +100 mV in 1 M KCl.Right, the occurrence rate constant of signature events for miR-155detection with different probes (Table 5). Significance (p<0.005) isvalid between results with any two probes; B. Left, [miR-155]-f₁₅₅correlation for target concentration ranging between 10-100 nM. Right,[miR-155]-f₁₅₅ correlation measured in 0.5 M/3 M (cis/trans) KClasymmetrical solutions for much lower target concentration between0.1-100 pM. Significance (p<0.01) was valid between detections at anytwo miR-155 concentrations.

FIG. 10 A-D shows differentiation of let-7 miRNAs containing one or twodifferent nucleotides. The sequence of let-7a, -7b, and -7c were givenin Table 3). a. Detections of let-7a and let-7b using the probe P_(a) orP_(b) at +120 mV. Left, current traces, and right, comparison ofsignature event duration (τ_(sig)); b. Detections of let-7a and -7cusing the probe P_(a) or P_(c) at +100 mV. Left, current traces, andright, comparison of signature event duration (τ_(sig)). Data was shownin Table 6; c. Receiver Operating Characteristic (ROC) curves fordiscrimination of events for miRNA·probe hybrids without (positive) andwith mismatches (negative). □: let-7a·P_(a)/let-7b·P_(a), ◯:let-7b·P_(b)/let-7a·P_(b), ▪: let-7a·P_(a)/let-7c·P_(a), and :let-7c·P_(c)/let-7a·P_(c); d. Correlation between the areas under theROC curves (AUC) and the duration ratio between fully-match events andmismatch events. : AUC measured from the ROC curves in panel c (Table7), ◯: AUC calculated from ROC analysis based on simulated datasets(FIG. 16 and Table 8). The events were generated with an exponentiallydistributed duration. The duration ratios of fully-match events(positive) and mismatch events (negative) were 1, 2, 3, 4, 5 and 10respectively.

FIG. 11 A-H shows detection of miR-155 in lung cancer patients' plasma.a through d. Signature events found in current traces for total plasmaRNAs from normal volunteers (a) and lung cancer patients (b) in thepresence of 100 nM P₁₅₅ probe, no signature events observed in theabsence of P₁₅₅ (c and d). The traces were recorded in 1 M KCl at +100mV. e. Frequencies of miR-155 signature events (f₁₅₅) from six normalindividuals (#1 to #6) and six patients with lung cancer (#7 to #12) inthe presence of spiked-in synthetic miR-39. f. Frequencies of miR-39signature events detected by P₃₉ (see the sequence in Table 3) from allsample used in e. Each sample was measured n times (n≧4) withindependent nanopores. The data was given as mean±SD. The patientconditions were, #7, metastatic squamous lung carcinoma; #8, recurrentsmall-cell cancer; #9, early stage of small-cell carcinoma, status postchemotherapy and radiation; #10, early stage of small-cell cancer,status post chemotherapy; #11, late stage non-small cell carcinoma,statue post resection and chemotherapy; #12, late stage adenocarcinoma,status post chemotherapy. g. f₁₅₅/f39 calculated from panel e and f. h.Box and Whiskers plot of relative miR-155 level in normal and lungcancer groups, measured with the nanopore sensor and qRT-PCR. The boxesmark the interval between 25^(th) and 75^(th) percentiles. The blacklines inside the boxes denote the medians. The whiskers denote theinterval between the 5^(th) and 95^(th) percentiles. Filled circlesindicate data points outside the 5^(th) and 95^(th) percentiles. Datawere given in Table 9.

FIG. 12 A-D shows histograms of block durations. a. Signature blocksgenerated by the mir-155·P₁₅₅ hybrid. b. The short Level 3 state in thesignature block. c. and d. Short blocks by translocation of miR-155 (c)and P₁₅₅ (d) alone. Data was obtained from current traces recorded in 1M KCl at +100 mV.

FIG. 13 shows voltage-dependent frequency of mir-155·P₁₅₅ signatureevents. Data was obtained from current traces recorded in 1 M KCl with10 (Δ) and 25 (□) nM mir-155 in the presence of 100 nM P₁₅₅, and 10 pMmir-155 in the presence of 5 pM P₁₅₅ (◯)

FIG. 14 shows the frequency of miR-155 signature events detected usingP₁₅₅(100 nM) in the presence of other synthetic miRNA components. Thethree bars represented miR-155 alone (50 nM), miR-155 in the presence ofLet-7a (50 nM), and that in the presence of both Let-7a (50 nM) and -7b(50 nM). Data was obtained from current traces recorded in 1 M KCl at+100 mV.

FIG. 15 shows duration histograms of signature events formed byLet-7a·P_(a) and Let-7b·P_(a) hybrids. Data was obtained from currenttraces recorded in 1 M KCl at +120 mV. a. Let-7a·P_(a). b. Let-7b·P_(a).Concentrations of all RNA and DNA components were 100 nM.

FIG. 16 A-B shows simulation on separation of fully-match (positive) andmismatch (negative) events based on event duration. a. ROC curves atvarious duration ratios. There were 400 events of both typesparticipating in the analysis; b. ROC curves at various event numberratios of the two type of events. The duration ratio τ_(P)/τ_(N)=3.

FIG. 17 shows translocation frequencies of miR-155 and P₁₅₅. Data wasobtained from current traces recorded in 1 M KCl at +100 mV. Theconcentrations of both oligos were 100 nM.

FIG. 18 A-F shows: A) the diagram of the miRNA/probe complex. FIG. 18 Bshows events for translocation of the peptide-PNA probe, P7b. Thecharacteristic events last for 3 ms and reduce the current to 10 pA at+180 mV. FIG. 18C shows that no block events can be observed with freemiRNA let-7b (without probe) in the solution at +180 mV. FIG. 18D showssignature events for the trapping of the let-7b/P7b complex. FIG. 18Eshows that Let-7c, which has two different nucleotides from Let-7b,cannot bind to PNA of the probe P7b, therefore does not generatesignature events as in FIG. A1c. Almost all observed events are due tothe probe itself. FIG. 18F compares the duration-amplitude property forP7b binding to Let-7b (fully match, two separate clusters withoutoverlay) and Let-7c (2 mismatches, two clusters fully overlay).

FIG. 19 A-C shows: FIG. 19A, when employing HP-C30 with a hairpin at the3′-end of short strand, we observed a novel type of three-level currentpattern. FIG. 19B, when using SA-C30 attached with a streptavidin at the3′-end of the short strand, we also observed a new multi-level currentpattern. FIG. 19C shows when using a short oligonucleotide to link twoDNAs, the complex can be sequentially unzipped in the nanopore in twosteps. The two unzipping can be clearly revealed by the two Level 2states.

DETAILED DESCRIPTION OF INVENTION

The invention provides a robust nanopore sensing system that enablessensitive, selective and direct detection, differentiation andquantification of single strand oligonucleotide, such as miRNAs.Additionally, the inventive sensing technology can also be employed todiscriminate single nucleotide differences in miRNA family members.Furthermore, the inventive technology has the potential for non-invasiveand cost-effective early diagnosis and continuous monitoring of cancermarkers in patients' blood samples.

The inventive nanopore sensing system for detecting a targetsingle-strand oligonucleotide, such as a miRNA, includes 1) a nanoporeallowing rapid translocation of single-stranded oligonucleotide, 2) apower source providing a pre-determined voltage as driving force toinduce unzipping of a double-stranded oligonucleotide, 3) a probemolecule to be mixed with the target oligonucleotide and loaded into thenanopore, and the unzipping of the hybrid of target oligonucleotide andthe probe in the pore produces certain identifiable current signalchanges, and 4) a means for detecting current changes.

Refer to FIG. 1, which is a schematic illustration of an exemplarynanopore sensing system. As shown in FIG. 1, the sensing chamber, 1,includes a cis compartment, 2, and a trans compartment, 3, which aredivided by a partition, 4. Both compartments are filled with apre-selected recording solution such as 1 M KCl. The partition, 4, hasan opening, 5, in its center region, over which a lipid bilayer isformed, and the nanopore, 6, is plugged through the lipid bilayer. Thepower, 7, provides a voltage that is loaded through a pair of electrodesin the two compartments; the current detector, such as a pico-Ampereamplifier, 8, is connected to monitor the current changes. Upon thetesting, a mixture sample of the target oligonucleotide, 9, and itscomplementary probe, 10, is loaded into the cis compartment, 2.

Refer to FIG. 2, which is a schematic amplified illustration of thenanopore, 6. As shown in FIG. 2, the nanopore, 6, is in conical orfunnel shape with two openings, the cis opening, 11, at the wide end andthe trans opening, 12, down the narrow end. During the detection, thepaired oligonucleotides, 9/10, is captured into the nanocavity, 13. Thevoltage then drive the oligonucleotides, 9/10, to unzip at theconstriction, 14, with the probe, 10, first traversing through theβ-barrel, 15, and out off the trans opening, 12, and followed by thetraversing of the target oligonucleotide, 9.

The nanopore may be any ion channel of cone-shape or any asymmetricalshape with a wide and a narrow opening plugged into the planar lipidbilayer that has a wider cavity followed by a narrow channel that canfacilitate unzipping translocation events. The nanopore may be anyexisting protein ion channels, such as the α-hemolysin transmembraneprotein pore adopted in the examples below, or various synthetic poresfabricated using fashion nanotechnologies with abiotic materials such assilicon.

The inventive probe is a multi-domain single strand molecule, whichcomprises a central domain fully complementary to the targetoligonucleotide and at least one terminal extension, i.e., signal tag,at its 3′ or 5′ terminal, with signal tags at both terminals aspreferred. The invention suggests the 3′-tagged probe is preferred overthe 5′-tagged probe. The probe directionality-dependence of the capturerate is possibly due to that the bases of ssDNA tilt collectively towardthe 5′ end of the strand³⁸, and this asymmetric base orientation makesDNA move more easily from 3′-end than 5′-end.

The terminal extension (signal tag) may be of any charged single chainmolecule with sufficient length to assist the unzipping translocationthrough the nanopore driven by the voltage. The signal tag may be acharged polymer chain, which can be an oligonucleotide such aspoly(dC)_(n), poly(dA)_(n), and or poly(dT)_(n), or a chargedpolypeptide. For example, when α-hemolysin transmembrane protein pore isemployed as the nanopore, the poly(dC) tag is more preferred overpoly(dA) or poly(dT) tags; furthermore, the poly(dC)₃₀ is much moreefficient in generating signature events (discussed below) than thatwith a shorter tag such as poly(dC)₈. The capture rate can be furtherenhanced once combined with other effective approaches, includingdetection at high voltage, use of engineered pores with designed chargeprofile in the lumen³³, and detection in asymmetrical saltconcentrations between both sides of the pore³⁹.

The invention also provides a method of detecting and differentiatingsingle strand oligonucleotides by monitoring the current changes inducedby the unzipping and translocation of the oligonucleotides through ananopore. The inventive method of detecting single strandoligonucleotide with a dual-compartment nanopore system, as the oneillustrated in FIG. 1, includes the steps of 1) mixing the targetoligonucleotide with a pre-designed probe, which has its central domainmatching the target sequence and a charged single chain molecule taggedto at least one of its 3′ and 5′ terminals, to produce a sample mixture,2) loading the mixture into the cis compartment, 3) providing the systemwith a pre-determined voltage, and 4) recording current output for apre-determined time period. The current change induced by the unzippingand translocation of the hybrid of the target oligonucleotide and itscomplementary probe through the nanopore is a unique signature event,which is used to detect and differentiate the target oligonucleotide.Refer to FIG. 3, which includes an exemplary current trace recordedduring an exemplary detection, an amplified electrical mark of thesignature event, and a schematic illustration of theunzipping-translocation event.

Further Description

Definitions

As used herein, the term “ROC curve” refers to a Receiver OperatingCharacteristic Curve. An ROC curve used to analyze the relationshipbetween selectivity and sensitivity. An ROC curve separates the plotinto up and lower regions.

As used herein, the term “AUC” refers to the Area under the ROC curve.An AUC can range between 0.5-1.0. The higher the AUC value, the betterthe separation result.

As used herein, the term “OCP” refers to an Optimized Cutoff Point. Incertain embodiments, an OCP can be calculated from ROC curves. Incertain embodiments, an OCP is a cutoff duration at the maximal value ofa Youden index.

As used herein, the phrase “Youden index” is defined as{sensitivity+selectivity−1}. A Youden index is calculated from the ROCcurve, and can range between 0 and 1. A cutoff duration leading tocomplete separation of long and short duration distribution results inYouden index=1, whereas complete overlap gives Youden index=0. Incertain embodiments, a cutoff duration value that returns the maximum ofYouden index, i.e. “optimal” cutoff point (OCP) (Greiner et al., 2000Preventive Veterinary Medicine 45, 23-41) gives the most accurateseparation.

Description of the Probes, Nanopores, Kits Comprising the Probes andNanopores, and Associated Methods of Use

In one broad aspect, the instant invention is directed to probes,nanopores, kits comprising the probes and nanopores, and associatedmethods of use, that provide for “signature” current blockage eventsthat distinguish those events arising from interactions with the probeand target from other events. In this context, the other events arereferred to as “background” events. Background events include, but arenot limited to, interactions of a probe with nucleic acid that is not atarget, interactions of a probe with other components present in ananopore detection system, free nucleic acids present in the nanoporedetection system, and the like. Such features of such signature eventsinclude, but are not limited to, at least one of a: i) a current blockof different duration than a background current block; ii) a differentnumber of distinct current blockade levels than a background currentblock; iii) a different order of occurrence of current blockade levelsthan a background current block; iv) a different current amplitude at ablockade level than a background current block; v) a different currentamplitude of each blockade level than a background current block; or anycombination of (i), (ii), (iii), (iv), or (v). In certain embodiments, asignature blockage event can be distinguished from a background blockageevent by differences in a characteristic background noise of eachblockage event. In certain embodiments, the distinct durations, numbers,or amplitude(s) in the signature event are greater than those observedin the background event. In certain embodiments, the distinct durations,numbers, or amplitude(s) in the signature event are less than thoseobserved in the background event. In certain embodiments, the distinctdurations, numbers, orders, or amplitude(s) in a signature event arestatistically distinguishable from those of a background event. Incertain embodiments, the signature events are provided in nanoporesystems comprising a protein nanopore formed by alpha-hemolysin (αHL) orengineered variants thereof in a planar lipid bilayer system. In certainembodiments, the signature events can be provided in a biochip formed byhydrogel-encapsulated lipid bilayer with a single protein nanoporeembedded therein or a micro-droplet bilayer system. Biochips andmicro-droplet bilayer systems have been described (Shim and Gu;Stochastic Sensing on a Modular Chip Containing a Single-Ion ChannelAnal. Chem. 2007, 79, 2207-2213; Bayley, H. et al. Droplet interfacebilayers. Mol. Biosyst. 4, 1191-1208 (2008).

In certain embodiments, the signature events can be provided in asynthetic nanopore. Synthetic nanopores include, but are not limited to,nanopores comprising silicon nitride or graphene.

Probe molecules provided herein comprise terminal extensions at one orboth of their 5′ and/or 3′ termini. Without seeking to be limited bytheory, it is believed that these terminal extensions provide usefulfunctions that include, but are not limited to, trapping of theprobe/target complex into the nanopore at a high rate (i.e. the numberof signature events per unit target concentration per unit recordingtime). The trapping rate directly determines the sensitivity. In thesame target concentration and the same recording time, a higher trappingrate gives a more precise sensing result. Without seeking to be limitedby theory, it is also believed that these terminal extensions provideuseful functions that include, but are not limited to, inducing thevoltage-driven dissociation of the probe/target complex. Thisdissociation function generates a signature event that can be used todiscriminate interactions of the probe with the target from othercomponents in the mixture, thereby ensuring the selectivity orspecificity.

Probe terminal extensions can comprise a charged polymer of any length.In certain embodiments, the polymer can be a negatively chargedsingle-stranded nucleic acid. Advantages of such nucleic acid terminalextensions include, but are not limited to, extremely low cost ofsynthesis and controllable charge by pH, salt concentration andtemperature. Such nucleic acid extensions can comprise homopolymers,heteropolymers, copolymers or combinations thereof. In certainembodiments, the lengths of such nucleic acid terminal extensions canrange from about 1 or 2 nucleotides to about 50 nucleotides. In stillother embodiments, the nucleic acid extensions can range in length fromabout 5 to about 40 nucleotides, about 15 to about 35 nucleotides, orfrom about 20 to about 35 nucleotides. An exemplary terminal extensionprovided herewith is homopolymer poly(dC)₃₀. However, a heteropolymericsequence, including but not limited to, di- or tri-nucleotideheteropolymers such as CTCTCTCT . . . , or CATCATCAT . . . , can also beused. In certain embodiments, co-polymers comprising abases orpolyethylene glycol (PEG) can be used in the terminal extension. Theseco-polymers, or domains thereof in a terminal extension, can confer newfunctions on the terminal extension of the probe. An abase is anucleotide without the base, but carries a negative charge provided bythe phosphate. As the dimension of abase is narrower than normalnucleotides, it may generate a signature event signal different fromthat formed by the neighbor nucleotides. PEG is not charged. Withoutseeking to be limited by theory, it is believed that when the PEG domainin a nucleic acid sequence is trapped in the pore, it can reduce thedriving force, thus precisely regulating the dissociation of theprobe/target complex.

Probe terminal extensions can also comprise a polypeptide. The richerchoice of amino acids makes the sequence and functionality of thepolypeptide terminal extension more programmable than an oligonucleotideterminal extension. For example, polypeptide terminal extensions allowinsertion of charged amino acids in the optimized positions to generatemore distinguishable probe/target signature events. While not seeking tobe limited by theory, it is believed that the probe/target complex canbe selectively trapped using a probe comprising a positively chargedpolypeptide terminal extension under an appropriate voltage while allother negatively charged non-target oligonucleotides in the mixture areprevented from entering into the pore, resulting in ultra-selectivedetection. In certain embodiments, the polypeptide terminal extensionscan comprise two, three, four, or more amino acid residues that cancarry a positive charge (i.e. lysine and/or arginine and/or histidine).In certain embodiments, sufficient numbers of positively chargedresidues are included in the polypeptide terminal extension to provide anet positive charge when said probe is hybridized to a targetoligonucleotide. In certain embodiments where probes comprising terminalextensions with positive charges conferred by residues such as lysine,arginine or histidine, performance of the associated nanopore baseddetection methods can be enhanced under acidic conditions (i.e. when thepH value is less than 7) or conditions where the residue will beprotonated. Thus, the use of such probes at pH values of about 1 toabout 6.9, 1 to about 6.0, about 1 to about 5.5, about 3 to about 5.5,and the like. In certain embodiments, the lengths of such polypeptideterminal extensions can range from about 1 or 2 residues to about 30residues. In still other embodiments, the polypeptide extensions canrange in length from about 5 to about 20 residues, about 8 to about 20residues, or from about 8 to about 15 residues. In an exemplaryembodiment, an HIV-TAT polypeptide comprising positively chargedarginine and lysine residues can be used as the terminal extension. Incertain embodiments, the center domain of the probe that iscomplementary to the target oligonucleotide can comprise a peptidenucleic acid that is covalently linked to a terminal extensioncomprising amino acids that carry a positive charge. In certainembodiments, a center domain comprising a peptide nucleic acid is usedin conjunction with a terminal extension comprising amino acids thatcarry a positive charge to provide a net positive charge when said probeis hybridized to a target oligonucleotide. In certain embodiments,polypeptide terminal extensions comprising amino acids with aromaticside chains including, but not limited to, phenylalanine, tryptophan,tyrosine, thyroxine, and the like, can be incorporated into thepolypeptide terminal extensions. While not seeking to be limited bytheory, it is believed that such aromatic amino acids can interact withthe pore through aromatic stacking and provide for useful changes in thesignature obtained in nanopore based detection methods.

Without seeking to be limited by theory, it is believed that if thereare a sufficient number of positively-charged amino acids in thepolypeptide terminal extension such that the net charges of the targetoligonucleotide/probe complex are still positive when the probecomprising the terminal extension is hybridized to the targetoligonucleotide, then the entire target oligonucleotide/probe complexwill form a strong dipole molecule. It is believed that thepositively-charged peptide domain of the probe dipole will be bothpushed by the positive voltage (cis grounded) and attracted by thenegative ring at the trans opening, guiding the trapping of theoligonucleotide/probe complex into the pore. At the positive voltage,any other free nucleic acids components will be repulsed from enteringthe pore due to the negative charge that is carried by the free,unhybridized nucleic acids. This significantly reduces signals by freenucleic acid components, such that the majority of the observed currentblockage events are either due to the trapping of theoligonucleotide/probe complex or to the translocation of the probe.

In certain embodiments, the oligonucleotide/probe complexes with a netpositive charge can be directed to a nanopore with a negatively-chargedring at the trans- opening of the pore. In this context, a trans openingof a pore is understood to be that portion of the pore from which amolecule would emerge whereas a cis opening of a pore from which amolecule would enter. In these embodiments, it is understood that anegative charged ring at the trans- opening of the pore can be obtainedby using any type of nanopore that has been suitably synthesize and/orderivatized so as to have a negative charged ring at the trans- openingof the pore. Such nanopores with a negatively charged ring at the transopening of the pore include, but are not limited to, protein nanoporesand synthetic nanopores. Protein nanopores with a negatively chargedring at the trans opening of the pore include, but are not limited to,engineered variants of an alpha-hemolysin protein. In certainembodiments, the engineered alpha hemolysin variant can comprise aStaphylococcus aureus alpha hemolysin containing a K131D, a K131E, or aK131H amino acid substitution. Exemplary and non-limiting Staphylococcusaureus alpha hemolysin wild type sequences are provided herein (SEQ IDNO:20, nucleic acid coding region; SEQ ID NO:21: protein coding region)and available elsewhere (National Center for Bioinformatics or GenBankAccession Numbers M90536 and AAA26598). An exemplary and non-limitingStaphylococcus aureus alpha hemolysin variant comprising a K131Dsubstitution is provided as SEQ ID NO:22. In certain embodiments, theengineered alpha hemolysin variant can comprise a suitably derivatizedvariant that is derivatized with moieties that provide for a negativelycharged ring at the trans opening of the pore. An exemplary wild type S.aureus alpha hemolysin protein that can be substituted or derivatized toprovide for a protein nanopore with a negative charged ring at thetrans- opening of the pore is provided herewith as SEQ ID NO: 21.However, variants of other hemolysins capable of forming pores can besubstituted or derivatized to provide for a protein nanopore with anegative charged ring at the trans- opening of the pore. Syntheticnanopores with a negatively charged ring at the trans opening of thepore are also provided. In certain embodiments, such synthetic nanoporeswith a negatively charged ring at the trans opening of the pore include,but are not limited to, silicon nitride or graphene nanopores that havebeen suitably derivatized with moieties that provide for a negativelycharged ring at the trans opening of the pore.

The center domain of probes provided herein is used to capture thetarget molecule. In certain embodiments, the center domain can be fullycomplementary or partially complementary to the target sequence. Incertain embodiments, a center domain can comprise an oligonucleotidecomprising natural nucleotides (A, T, G, C (DNA) or a, u, g, c (RNA)),and/or artificial nucleotides including, but not limited to, nucleosidessuch as inosine, xanthosine, 7-methylguanosine, Dihydrouridine, and5-methylcytidine. In certain embodiments, the center domain can comprisea locked nucleic acid (LNA) or a peptide nucleic acid (PNA). Lockednucleic acids comprise RNA derivatives where the ribose ring contains amethylene linkage between the 2′-oxygen and the 4′-carbon. Peptidenucleic acids (PNA) comprise a peptide backbone with nucleobase sidechains. In certain embodiments, a LNA or a PNA center domain cancomprise natural nucleobases (adenine, guanine, thymine, cytosine oruracil) and/or artificial nucleobases including, but not limited to,hypoxanthine, xanthosine, 7-methylguanine, 5,6-dihydrouracil, and5-methyl cytosine. In certain embodiments, probe center domainscomprising co-polymers of oligonucleotides, LNA, or PNA are provided. Incertain embodiments, a center domain of a probe will have at least about4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotide or nucleobase residues that are complementary to the targetnucleic acid. In certain embodiments, a central region of a probe willhave at least about 4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 to any of about 30, 35, 40, or 50 nucleotide or nucleobaseresidues that are complementary to the target nucleic acid. In certainembodiments, synthetic nucleotides or nucleobases inserted in thesequence can precisely adjust the hybridization energy with the target,such that one can distinguish the characters of targets such assingle-nucleotide polymorphism, methylation, or interaction betweenmiRNA and its target messenger RNA.

A variety or target nucleic acids or oligonucleotides that can bedetected and distinguished from non-target nucleic acids by the probes,nanopores, kits comprising the probes and nanopores, and associatedmethods of use probes, provided herein. In certain embodiments, thetarget can be a nucleic acid or a fragment thereof from cells, bodyfluid, tissues, bacteria, or a virus. In certain embodiments, the targetcan be a PCR products or a synthetic oligonucleotide. In certainembodiments, a target can comprise a genomic DNA, an mRNA , a pre-matureor mature miRNA, an artificial miRNA, non-coding DNA or RNA, a nucleicacid biomarker, or a synthetic aptamer. In certain embodiments, a miRNAtargets may come from the RNA extraction from bio-fluid from any tissuessuch as plasma and formalin-fixed and paraffin-embedded tissues. Incertain embodiments, a target nucleic acid can comprise be a nucleicacid fragment complexed with any of a nucleic acid binding protein, anantibody, or an aptamer bound with a target protein. In certainembodiments, a target nucleic acid can comprise be a nucleic acidfragment complexed with a low molecule weight compound, including, butnot limited to, a drug. In certain embodiments, targets can includesequences with mutations, with single-nucleotide polymorphisms, or withchemical modifications such as methylation and phosphorylation.

EXAMPLES Example 1 Detection of miR-155, a Lung Cancer Biomarker

The invention further provides an exemplary nanopore sensing system fordetection of miR-155, a lung cancer biomarker. The nanopore sensingsystem includes an α-hemolysin transmembrane protein pore and apre-designed probe for miR-155. The probe is a DNA multiple-blockcopolymer with its central domain complementary to the target miR-155,and at least one poly(dC)₃₀ extension at 3′- , 5′-, or both terminalsfunctioning as signal tags. Table 1 lists the sequences of miR-155 andthe exemplary probes with the tri-block copolymer, P₁₅₅ as preferred.

TABLE 1 Sequences of miRNAs and their probes miRNA Probe Sequencemir-155 5′-UUAAUGCUAAUCGUGAUAGGGG-3′ (SEQ ID NO: 1) P_(nt)5′-CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 2) P_(5′-C30)5′-C₃₀-CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 3) P_(3′-C30)5′-CCCCTATCACGATTAGCATTAA-C₃₀-3′ (SEQ ID NO: 4) P₁₅₅5′-C₃₀-CCCCTATCACGATTAGCATTAA-C₃₀-3′ (SEQ ID NO: 5)

During an exemplary sensing process, a mixture of miR-155/P₁₅₅ is addedto the cis side of the pore, a current trace with a series of short- andlong-lived current blocks can be recorded, as shown in FIG. 4 a, whilebeing monitored in 1 M KCl at +100 mV. The spike-like short blocks,labeled as b in FIG. 4 a and also shown in FIG. 4 b, have duration of220±21 μs and almost fully reduce the pore conductance with a relativeconductance g/g₀=0.16, where g and g₀ are conductance of blocks andunoccupied nanopore. Both the duration and conductance are similar tothat of blocks by miR-155 and P₁₅₅ alone, thus the short blocks in themixture are associated with the rapid passage of single-stranded freemiR-155 or P₁₅₅ through the pore, as illustrated in FIG. 4 b′.

In contrast to short blocks, the long blocks, labeled as c and d in FIG.4 a, in the recording persist for 250±58 ms. One type of long blocks,labeled as c in FIG. 4 a, features three sequential conductance levels,Level 1→Level 2→Level 3 (expended in FIG. 4 c). This type of blocks isnot observed when either miR-155 or P₁₅₅ alone is presented, indicatingthat it is originated from the miR-155/P₁₅₅ hybrid (miR-155·P₁₅₅). Level1 almost fully reduces the conductance to g/g₀=0.15. This conductancelevel is consistent with a configuration that miR-155·P₁₅₅ is trapped inthe pore from the wider opening (cis), with either 3′- or 5′-signal tagof P₁₅₅ occupying the narrowest β-barrel (FIG. 4 c′ level 1). The signaltag in the β-barrel can induce unzipping of miR-155·P₁₅₅, driven byvoltage. The unzipping time, or the duration of Level 1, is comparableto previously reported time scales for DNA unzipping in the pore, e.g.˜435 ms for unzipping a 50 bps dsDNA at +140 mV, and ˜40 ms for a 10 bpshairpin DNA at +90 mV. The unzipping process was further evidenced bythe discrete transition from Level 1 to Level 2. Level 2 lasts 410±20 μsand its conductance significantly increases to g/g₀=0.42 (FIG. 1 c′level 2). This partial block can not be interpreted as anoligonucleotide occupying in the β-barrel. It is very likely that, afterunzipping of miR-155·P₁₅₅ followed by translocation of P₁₅₅, mir-155 canbe temporarily confined in the nanocavity of the pore. It has beenverified that a single-stranded oligonucleotide residing in thenanocavity can generate such a partial block^(33,34). The miR-155molecule in the nanocavity finally traversed the β-barrel, generatingLevel 3 which fully reduces the conductance to g/g₀=0.08 (FIG. 4 c). Theduration of Level 3 is 270±30 μs, close to the 220 μs for short blocksby mir-155 alone, and consistent with the time scale of ˜400 ps fortranslocation of a 75 bases RNA at +120 mV, ³⁵ and 800 μs for a 210bases RNA at +120 mV.³⁶ The duration of Level 3 becomes shortened as thevoltage increases, further supporting the translocation of asingle-stranded oligonucleotide for this conductance level.

In addition to the multi-conductance long blocks, the long blocks withsingle conductance level at g/g₀=0.15 (labeled as d in FIG. 4 a andexpended in FIG. 4 d) have also been observed. This type of long blocksmay occur when the arrested miR-155·P₁₅₅ exits the pore from the cisentry without unzipping.

The invention teaches that the characteristic long blocks can serve assignature events for identifying single molecules of target miRNAs. Fromthe frequency of signature events (f_(sig)), the target miRNA can bequantified using Eq. 1 (Methods in Supplementary Materials, providedbelow in Example 2),

$\begin{matrix}{f_{sig} = {k_{on}\frac{\begin{matrix}{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right) -} \\\sqrt{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right)^{2} - {{4\lbrack{miR}\rbrack}_{0}\lbrack P\rbrack}_{0}}\end{matrix}}{2}}} & (1)\end{matrix}$

In Eq. 1, [miR]₀ and [P]₀ are the initial concentrations of miRNA andthe probe, k_(on) is the occurrence rate of signature events and K_(d)is the dissociation constant for miR·P in the solution. When[miR]₀<<[P]₀, f_(sig)≈k_(on)[miR]₀. The current traces indeed show morefrequent miR-155·P₁₅₅ signature events as the miR-155 concentrationincreases (FIG. 5 a). The f_(sig)-[miR-155] data can be best fittedusing Eq. 1, with K_(d)=30 nM and k_(on)=3.6×10⁶ M⁻¹s⁻¹ (FIG. 5 b).According to the literature⁸, the mean concentrations of circulatingmiRNAs were 158.6 ng/mL (˜25 nM) for the lung cancer group versus 68.1ng/mL (˜10 nM) for the control group. Therefore we compared the f_(sig)values at 10 nM and 25 nM mir-155 (FIG. 5 b). Analysis indicates thatthe two levels of miRNA concentration can be separated (p<0.005),suggesting that the inventive method has the potential to differentiallydetect miRNA levels in lung cancer patients.

The invention also provides an exemplary process employing the inventivenanopore sensing system to differentiate highly similar miRNA sequences,let-7a and let-7b. let-7a and let-7b are members of the Let-7 tumorsuppressing miRNA family⁴⁻⁶; and the two Let-7 members only containdifferent nucleotides at the position 17 and 19, which are adenines inlet-7a and guanines in let-7b. The inventive probes P_(a) and P_(b) aredesigned for let-7a and let-7b respectively with sequences listed inTable 2.

TABLE 2 Sequences of let-7a and let-7b and their probes miRNA ProbeSequence Let-7a 5′-UGAGGUAGUAGGUUGU A U A GUU-3′ (SEQ ID NO: 6) P_(a)5′-C₃₀-AACTATACAACCTACTACCTCA-C₃₀-3′ (SEQ ID NO: 7) Let-7b5′-UGAGGUAGUAGGUUGU G U G GUU-3′ (SEQ ID NO: 8) P_(b)5′-C₃₀-AACCACACAACCTACTACCTCA-C₃₀-3′ (SEQ ID NO: 9)

As shown in FIG. 6 a and FIG. 6 b, when using P_(a) to detect eachmiRNA, the duration of signature events (τ_(sig)) for let-7a·P_(a)(without mismatch) is 155±28 ms, whereas τ_(sig) for let-7b·P_(a) (with2-nt mismatches) is significantly shortened to 48±11 ms (p<0.005).Similarly, when using P_(b) to detect both miRNAs, τ_(sig) forlet-7b·P_(b) (without mismatch) is 165±47 ms, significantly longer thanthe 24±2 ms for let-7a·P_(b) (with 2-nt mismatches) (p<0.005) as shownin FIG. 6 b. The significant differences in durations can be interpretedthat the mismatches significantly weaken the hybridization interactionbetween miRNA and the probe. When placed in the identical electricalfield, the hybrid containing mismatches needs lower energy than fullymatched hybrid to unzip. Thus, the inventive nanopore sensing system isable to differentiate single mismatches based on the unzipping time,thus demonstrating the potential to detect miRNAs with similar sequencesand SNPs.

The invention further provides an exemplary process of detecting plasmamiR-155 in lung cancer patients with the inventive nanopore sensingsystem. During the exemplary process, the peripheral blood samples wereobtained from six lung cancer patients and six normal volunteers with alocal IRB approval. Total plasma RNAs containing miRNAs were extractedfrom 350 μl of each plasma sample using miRVana PARIS Kit (Ambion), witha final elution volume of 100 μl, which were than divided into twoaliquots (50 μl ) for the nanopore and RT-PCR assay⁴⁴. One aliquot waspre-mixed with P₁₅₅ and directly added to the 2-ml recording solution inthe nanopore chamber. The nanopore current retain a low level of noiseeven in the presence of plasma samples, and distinct short and longblocks (marked with red arrows) can be indentified in both the controlgroup (FIG. 7 a) and lung cancer group (FIG. 7 b). The characteristiclong blocks, including both with multiple conductance and singleconductance, features the same conductance profiles and similarproperties to that for synthetic miR-155 RNA in FIG. 1 a. In the absenceof P₁₅₅, no such types of long blocks can be observed (FIGS. 7 c and d),but short blocks were found for translocation of single-strandedoligonucleotides such as free miRNAs (FIGS. 7 c and d). Overall, thecharacteristic long blocks could be attributed to miR-155·P₁₅₅ hybridsand serve as signature events for single miRNA molecules detection.

The frequency of miR-155 signature events f_(sig) for all samples in thelung cancer patient group varies between 1.15-1.51 min⁻¹, with a mean of1.40±0.16 min⁻¹ (FIG. 7 e). This level was significantly higher thanf_(sig) in the control group that ranges between 0.32-0.70 min⁻¹ with amean of 0.48±0.14 min⁻¹ (FIG. 7 e). Since all samples were preparedfollowing a standard procedure (Methods in Supplementary Materials), itshould be valid to compare relative miRNA levels in two groups. When themean f_(sig) value in normal plasma was set as 1, the folds of miR-155in lung cancer plasma were compared with the two methods. FIG. 7 fshowed that the relative mir-155 level in lung cancer patients was 2.79with the nanopore sensor (p<0.001). By comparison, the relative miR-155level was 4.72 with RT-PCR method (p<0.02) with greater variability.Therefore, both nanopore and RT-PCR assay indicated a significantelevation of miR-155 in lung cancer patient plasma although there is a1.69 fold difference. As the nanopore method does not require labelingand amplification, this may be a reason for smaller variability in thenanopore assay (FIG. 7 f). Overall the nanopore sensor with engineeredprobes demonstrates the ability to detect circulating miRNAs in clinicallung cancer patients, which is verified by the independent RT-PCRmethod.

Example 2 Supplementary Information

Materials. Oligonucleotides including miRNAs and DNA probes weresynthesized and electrophoresis-purified by Integrated DNA Technologies(Coralville, Iowa). Before testing, the mixtures of miRNA and DNA probewere heated to 90° C. for 5 minutes, then gradually cooled down to roomtemperature and stored at 4° C. The RNase-free water was used to prepareRNA solution.

Setup and method of nanopore detection. This section has beenwell-documented earlier (Shim, J. W., Tan, Q., & Gu, L. Q.Single-molecule detection of folding and unfolding of a singleG-quadruplex aptamer in a nanopore nanocavity. Nucleic Acids Res. 37,972-982 (2009)). Briefly, the recording apparatus was composed of twochambers (cis and trans) that were partitioned with a Teflon film. Theplanar lipid bilayer of 1,2-diphytanoyl-sn-glycerophosphatidylcholine(Avanti Polar Lipids) was formed spanning a 100-150 nm hole in thecenter of the partition. Both cis and trans chambers were filled withsymmetrical 1 M salt solutions (KCl) buffered with 10 mM Tris andtitrated to pH 8.0. All solutions are filtered before use. Singleα-hemolysin proteins were inserted into the bilayer from the cis side toform molecular pores in the membrane. All the oligonucleotides includingmiRNAs and DNA probes and clinical RNA samples were also added to thecis solution. To record the pore current, the cis solution was groundedand the voltage was given from the trans solution. In this convention, apositive voltage can drive the translocation of a negatively charged DNAthrough the pore from cis to trans. Single-channel currents wererecorded with an Axopatch 200A amplifier (Molecular Device Inc.Sunnyvale, Calif.), filtered with a built-in 4-pole low-pass BesselFilter at 5 kHz, and acquired with Clampex 9.0 software (MolecularDevice Inc.) through a Digidata 1332 A/D converter (Molecular DeviceInc.) at a sampling rate of 20 kHz. The data were analyzed usingClampfit 9.0 (Molecular Device Inc.), Excel (MicroSoft) and SigmaPlot(SPSS) software.

The translocation of free miRNA or probe through the pore generated veryshort current block (˜10¹-10² μs). Some short blocks showed partiallyreduced pore conductance, which may be due to the filtering of therecording at 5 kHz, or formed by the trapped oligonucleotide returningback to the cis solution (Maglia, G., Restrepo, M. R., Mikhailova, E., &Bayley, H. Enhanced translocation of single DNA molecules through +/−hemolysin nanopores by manipulation of internal charge. Proc. Natl.Acad. Sci. U.S.A. 105, 19720-19725 (2008). In our experiments, all shortblocks including these partial blocks were collected for histogramconstruction. Since the translocation events (˜10¹-10² μs) are welldistinguished from the signature events (˜10¹-10³ ms), we simply used 1ms as the boundary for separation of short and long events. Data weregiven as the mean±SD, based on at least three separate experiments(n>3). In the t-test, p<0.05 was considered as a significant differencebetween two groups. The electrophysiology experiments were conducted at22±2° C.

Total RNA Extraction From Plasma and miRNA Quantification by qRT-PCR.

Peripheral blood samples were obtained at the University of MissouriEllis Fischel Cancer Center with an IRB approval. Whole blood with EDTApreservative was centrifuged at 1,600 g for 10 min at room temperatureand the plasma was transferred to new tubes. Total RNAs containingmiRNAs was extracted from 350 μl of plasma using miRVana PARIS Kit(Ambion, Austin, Tex., USA) according to the manufacturer's protocol.The final elution volume was 100 μl.

A SYBR green-based quantitative RT-PCR assay was employed for miRNAquantification. In brief, 10 μl of total RNA sample containing miRNAswas polyadenylated by poly(A) polymerase (Ambion) and reversetranscribed to cDNA using SuperScript III Reverse Transcriptase(Invitrogen) according to the manufacturer's instructions with a poly(T)adapter primer (⁵′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTTTTVN-3′;SEQ ID NO: 10)). Real-time PCR was performed using iQ SYBR GreenSupermix (Bio-Rad, Hercules, Calif., USA) with the miR-155 specificforward primer (5′- -TTAATGCTAATCGTGATAGGGGT-3′; SEQ ID NO:11) and thesequence complementary to the poly(T) adapter as the reverse primer(5′-GCGAGCACAGAATTAATACGAC-3′; SEQ ID NO:12) in iQ5 Real-time PCR system(Bio-Rad, USA). The PCR was carried out as follows: after initialdenaturation at 95° C. for 3 min, 40 cycles of 95° C. for 15 s and 60°C. for 1 min were followed. The relative level of miR-155 was calculatedusing 2^(-delta Ct) method where the level of normal plasma wasnormalized as 1. Data was presented as mean±SD of three independentexperiments, and the differences were considered statisticallysignificant at p<0.05 by using the Student's t-test.

Normalization of the nanopore and qRT-PCR data using spiked-in C.elegans miRNA miR-39 as control. We introduced spiked-in synthetic miRNAas control, to convincingly validate the nanopore sensor's capability ofmiRNAs detection in human samples. The spiked-in RNA oligonucleotide inthe detection matches the sequence of C. elegans miR-39, a miRNA that isabsent in the human genomes. 3.5 μL, of 1 nM synthetic miR-39 solutionwas introduced to each 350 μL plasma sample after addition of the 2×Denaturing Solution (miRVana PARIS Kit) to the plasma, thus the miR-39concentration in plasma was 10 pM. The Denaturing Solution prevents RNAsfrom undergoing degradation by inhibiting endogenous plasma RNAases. Foreach sample, both miR-155 and spiked-in miR-39 were measured using thenanopore sensor and SYBR green-based qRT-PCR. The nanopore data andnormalization result were shown in Table 9. In the nanopore detection,the probes for miR-155 and miR-39 were P₁₅₅ and P₃₉. We first measuredthe signature event frequencies, f₁₅₅ and f₃₉, of the hybridsmiR-155·P₁₅₅ and miR-39·P₃₉ respectively. The variability of f₃₉reflected the difference in miR-39 concentrations among samples afterRNA extraction. Therefore the ratio of the two frequencies, f₁₅₅/f₃₉,should principally eliminate this variability. Finally, we used the Meanf₁₅₅/f₃₉ of six normal samples (A_(normal)) as the standard, andcalculated each sample's relative miR-155 level by normalizing f₁₅₅/f₃₉to A_(normal), i.e. f₅₅/f₃₉/A_(normal).

Correlation between miRNA concentration and frequency of signatureevents. In the deduction, “miR” represents miRNA; “P”, probe; K_(d),equilibrium dissociation constant for miR·P; k_(on), occurrence rateconstant of miR·P signature events; and f_(sig), frequency of signatureevents. In the mixture of miRNA and probe, the equilibrium can beestablished the reactors miR and P and the product miR·P,

$\begin{matrix}{{{miR} + P}\underset{\leftarrow}{\overset{K_{d}}{\rightarrow}}{{miR} \cdot P}} & \left( {{Scheme}\mspace{20mu} 1} \right)\end{matrix}$

K_(d) is determined by

$\begin{matrix}{K_{d} = \frac{\left( {\lbrack{miR}\rbrack_{0} - \left\lbrack {{miR} \cdot P} \right\rbrack} \right)\left( {\lbrack P\rbrack_{0} - \left\lbrack {{miR} \cdot P} \right\rbrack} \right)}{\left\lbrack {{miR} \cdot P} \right\rbrack}} & ({S1})\end{matrix}$

where [miR]₀ and [P]₀are total concentrations of miRNA and the probe,and [miR·P] is the concentration of miR·P. Thus the relationship of[miR·] and [miR]₀ is

$\begin{matrix}{\left\lbrack {{miR} \cdot P} \right\rbrack = \frac{\begin{matrix}{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right) -} \\\sqrt{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right)^{2} - {4\lbrack{miR}\rbrack}_{0} + \lbrack P\rbrack_{0}}\end{matrix}}{2}} & ({S2})\end{matrix}$

The kinetics for trapping and unzipping of miR·P in the nanopore is

Because f_(sig) is linearly related to [miR·P],

f _(sig) =k _(on)[miR·P]  (S3)

from Eq. S2 and Eq. S3,

$\begin{matrix}{f_{sig} = {k_{on}\frac{\begin{matrix}{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right) +} \\\sqrt{\left( {\lbrack{miR}\rbrack_{0} + \lbrack P\rbrack_{0} + K_{d}} \right)^{2} - {{4\lbrack{miR}\rbrack}_{0}\lbrack P\rbrack}_{0}}\end{matrix}}{2}}} & ({S4})\end{matrix}$

Eq. S4 suggested that f_(sig) is not in exact proportion to [miR]₀, thetotal concentration of the target miRNA. However, when [miR]₀ isconsiderably smaller than [P]₀, which is the case in our miRNAdetection, Eq. S4 can be simplified as

f _(sig) ≈k _(on)[miR·P]  (S5)

In this condition, f_(sig) is proportional to [miR]₀. Eq. S4 alsosuggested that f_(sig) will ultimately become saturated. This is becausef_(sig) measures the capture frequency of miR·P, and the maximalconcentration of miR·P ([miR·P]) can not be higher than that of theprobe ([P]₀).

Identification of miR-155 in trans solutions using RT-PCR. As shown inthe model (FIG. 8), with the miRNA·probe complex unzipped in the pore,the separated probe and miRNA can sequentially translocate through theβ-barrel to the trans solution. To verify this model, we employed RT-PCRto detect the unzipped miRNAs in the trans solution. However, the PCRmethod cannot discriminate if the trans miRNAs are from the unzippedmiRNAs or from the free miRNA (un-hybridized) that simply translocatedfrom the cis solution to the trans solution. We therefore added a muchhigher concentration of the probe than miRNA in the cis solution, sothat most of the miRNAs molecules are bound with the probe and there islittle free miRNA left, eliminating the translocation of free miRNA tothe trans solution that can interfere with the PCR result. Our targetwas miR-155 and the probe was P_(155.) The target/probe concentrationswere 0.1/1000, 1/1000 and 10/1000 (nM). After over 6 hours bilayerrecording for many pores, 2 μL of both cis and trans solutions weresubjected to polyadenylation, reverse transcription and RT-PCR to detectthe concentration of miR-155 as indicated in the method described above.Meanwhile, a series of dilution of synthesized miR-155 were performed toconstruct standard curve for calibration. The cis and trans miR-155 weremeasured separately. In the case of 10/1000 (nM) miR-155/P₁₅₅concentrations, the peaks of melting curves for trans RNA samples werethe same as synthesized miR-155. According to the standard curve ofmiR-155, the miR-155 concentrations in trans solutions were 14, 34 and63 aM (10⁻⁸ M), indicating that a trace amount of miR-155 transported tothe trans side of the pore.

Note S1. Precursor miRNAs (pre-miRNAs) are stem-loop RNAs of ˜70nucleotides bearing the 2 nucleotides 3′-overhang as a signature ofRNase III-mediated cleavage (Lee, Y., Jeon, K., Lee, J. T., Kim, S., &Kim, V. N. MicroRNA maturation: stepwise processing and subcellularlocalization. EMBO J 21, 4663-4670 (2002)). It is not known whether theplasma total RNA extract contains pre-miRNAs. However, we have verifiedthat the capture rate for a miR·P is very low if the signal tag in theprobe is very short (FIG. 9, FIG. 16 and unpublished data). Therefore,we expected that, even though pre-miRNAs exist, the short overhang willprevent them from trapping in the nanopore.

Note S2. We also compared the time spent for analyzing miRNA viananopore and qRT-PCR (in Supplementary Information). Our RT-PCRdetection can test at most 16 samples at once, including triplicates foreach sample with and without spike-in. PCR takes about 5-6 hours,including 1 hour PolyA reaction, 1 hour reverse transcription, 2.5 hoursqPCR, plus 1 hour for sample addition. The nanopore method is labelfree, does not need amplification, and is selective for short nucleicacids fragments. But our current nanopore setup allows detecting onesample at once. The average recording time for miR-155 from human plasmasample was about 90 minutes, collecting ˜100 events. Therefore, highthrough-put nanopore methods need developing. This is feasible becauseboth the synthetic nanopore array [Advanced Materials 18, 3149-3153(2006)] and the protein pore-synthetic pore hybridized system [Nat.Nanotechnol. 5, 874-877 (2010)] have been reported

TABLE 3 Sequences of studied miRNAs and their probes mir-1555′-UUAAUGCUAAUCGUGAUAGGGG-3′ (SEQ ID NO: 1) P_(nt)5′-CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 2) P_(5′-C30)5′-C₃₀-CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 3) P_(3′-C30)5′-CCCCTATCACGATTAGCATTAA-C₃₀-3′ (SEQ ID NO: 4) P₁₅₅5′-C₃₀-CCCCTATCACGATTAGCATTAA-C₃₀-3′ (SEQ ID NO: 5) Let-7a5′-UGAGGUAGUAGGUUGU A U A GUU-3′ (SEQ ID NO: 6) P_(a)5′-C₃₀-AACTATACAACCTACTACCTCA-C₃₀-3′ (SEQ ID NO: 7) Let-7b5′-UGAGGUAGUAGGUUGU G U G GUU-3′ (SEQ ID NO: 8) P_(b)5′-C₃₀-AACCACACAACCTACTACCTCA-C₃₀-3′ (SEQ ID NO: 9) Let-7c5′-UGAGGUAGUAGGUUGU A U G GUU-3′ (SEQ ID NO: 13) P_(c)5′-C₃₀-AACCATACAACCTACTACCTCA-C₃₀-3′ (SEQ ID NO: 14) miR-395′-UCACCGGGUGUAAAUCAGCUUG-3′ (SEQ ID NO: 15) P₃₉5′-C₃₀-CAAGCTGATTTACACCCGGTGA-C₃₀-3′ (SEQ ID NO: 16)

TABLE 4 Conductance of signature events produced by the miR-155·P155hybrid and spike-like short blocks by the translocation of miR-155 orP155 alone Signature block (miR-155·P₁₅₅) Level 1 Level 2 Level 3 Shortevent g (pS) 203 585 110 227 g/g₀ ^(a) 0.15 0.42 0.08 0.16 ^(a)g₀, theconductance of unoccupied α-hemolysin pore. g₀ = 1380 pS at +100 mV in1M KCl (pH8.0).

TABLE 5 Frequencies and occurrence rate constants of signature eventsdetected with various probes Probe P_(nt) P_(5′-C30) P_(3′-C30) P₁₅₅f_(sig) (min⁻¹)^(a) 0.168 ± 0.042 0.408 ± 0.084 8.31 ± 2.1 11.9 ± 1.2 (n= 5) (n = 4) (n = 5) (n = 6) k_(on) (M⁻¹s⁻¹)^(b) 2.8 ± 0.6 × 10⁴ 6.8 ±1.3 × 10⁴ 1.4 ± 0.3 × 2.0 ± 10⁶ 0.2 × 10⁶ ^(a)Frequencies in thepresence of 100 nM miR-155 and the probe at +100 mV ^(b)Occurrence rateconstant from f_(sig)≈k_(on) [miR-155], where [miR-155] = 100 nM

TABLE 6 Durations of signature events for fully-matched miRNA•probehybrids and that with mismatches miRNA•Probe let-7a•P_(a) let-7b•P_(a)let-7b•P_(b) let-7a•P_(b) τ_(sig) (ms) 155 ± 28 48 ± 11 165 ± 47 24 ± 2at +120 mV (n = 7) (n = 7) (n = 6) (n = 5) p-value <0.005 <0.005miRNA•Probe let-7a•P_(a) let-7c•P_(a) let-7c•P_(c) let-7a•P_(c) τ_(sig)(ms) 303 ± 45 124 ± 39 343 ± 49 179 ± 38 at +100 mV (n = 6) (n = 4) (n =6) (n = 4) p-value <0.005 <0.05

TABLE 7 Areas under ROC curves (AUC) for separation of miRNAs with onenucleotide difference (let-7a and let-7c) and with two nucleotidedifference (let-7a and let-7b)a let-7a · P_(a) let-7b · P_(b) let-7a ·P_(a) let-7c · P_(c) let-7b · P_(a) 0.75 n.a. let-7c · P_(a) 0.73 n.a.let-7a · P_(b) n.a. 0.83 let-7a · P_(c) n.a. 0.71 ^(a)The receiveroperating characteristic (ROC) curve is a plot of the true positive rate(sensitivity) against the false positive rate (1-selectivity) for thedifferent possible cutoff points that separate the entire durationdistribution into the positive and negative components. In the miRNAdetection, the events for fully matched miRNA.probe hybrids were denotedas “positive”, and that for mismatched hybrids as “negative”. Theseparation accuracy was measured by the area under the ROC curve (AUC).An AUC of 1 represents a perfect separation; an area of 0.5 representsno separation ability. AUC was analyzed online using free software onthe world wide web (internet address)rad.jhmi.edu/jeng/javarad/roc/JROCFITi.html.

TABLE 8 Areas under ROC curves (AUC) and optimal cutoff point (OCP) atvarious duration ratio and event number ratio a τ_(P)/τ_(N) (s/s) ^(b)1/1 2/1 3/1 4/1 5/1 10/1 AUC 0.51 0.72 0.73 0.76 0.78 0.93 OCP n.a. 1.331.74 1.88 1.98 2.18 N_(P)/N_(N) ^(c) 200:800 200:400 200:200 200:150200:100 200:50 AUC 0.83 0.81 0.76 0.76 0.79 0.78 OCP 1.88 1.88 1.85 1.791.81 1.97 a: Both AUC and OCP were calculated from the ROC curves shownin FIG. 16A-B. OCP is a cutoff duration at the maximal value of Youdenindex. Youden index is defined as {sensitivity + selectivity − 1},calculated from the ROC curve, and range between 0 and 1. A cutoffduration leading to complete separation of long and short durationdistribution results in Youden index = 1, whereas complete overlap givesYouden index = 0. The cutoff duration value that returns the maximum ofYouden index, i.e. “optimal” cutoff point (OCP) (Greiner et al., 2000Preventive Veterinary Medicine 45, 23-41) gives the most accurateseparation. ^(b) τ_(P)/τ_(N): The duration ratio of the “positive” and“negative” datasets. Each dataset contained 200exponentially-distributed duration values. The dataset with a longermean duration was denoted as “positive”; the shorter one as “negative”.^(c) N_(P)/N_(N): The event number ratio in the “positive” (N_(P))versus the “negative” dataset. τ_(P)/τ_(N) was 5 in this simulation.

TABLE 9 Levels of miR-155 and spiked-in miR-39 in human plasma samplesdetected by the nanopore sensor

^(a) Relative miR-155 level was obtained by normalizing each sample'sf₁₅₅/f₃₉ to the mean f₁₅₅/f₃₉ of the normal samples 1-6, which was 0.239as highlighted in the table.

Example 3 Peptide-Guided Selective Detection of MicroRNAs

Rationales. Our long-term goal is developing the nanopore singlemolecule sensor for accurate detection of target miRNAs in the total RNAextraction from plasma or tissues. The RNA extraction contains numerousand complicated nucleic acids components, at least including miRNAs(both pre-mature and mature miRNAs), mRNAs, tRNAs, other RNAs. Allcomponents commonly carry negative charges. Thus, if the target miRNAcan be trapped in the pore at an applied voltage, any other componentmay also be driven to interact with the nanopore, generatingnon-specific current signals that interfere with the recognition ofsignature events generated by the target miRNA/probe complex. Here weprovide a robust strategy for which only the target miRNA/probe complexcan be trapped in the pore, but any other nucleic acids components areprevented from interacting with the pore, therefore greatly improve theboth selectivity and sensitivity. This strategy is called peptide-guidedselective detection of miRNAs.

Methods. We designed a peptide-PNA (peptide nucleic acid) co-polymer asthe probe, as shown in FIG. 18 A. The PNA sequence (in “PNA” bracket inFIG. 18A) has a peptide backbone with side chain nucleobases that arecomplementary to the entire or partial sequence of the target miRNA(“miRNA (Let-7b, -7c)” in FIG. 18A bracket), and thus serves as thecenter domain for capturing the target miRNA in the solution. Thesesequences are provided as follows:

Probe

NH2-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-AACCACACAA-COOH, wherethe entire probe has a peptide backbone (i.e. the AACCACACAA portion ofthe probe comprises a peptide backbone with the indicated AACCACACAAnucleobases); SEQ ID NO:17.

PNA (Center Domain) of Probe:

NH2-AACCACACAA-COOH, where the molecule comprises a peptide backbonewith the indicated AACCACACAA nucleobases; (SEQ ID NO: 18).

HIV-TAT: (SEQ ID NO: 19) Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg

As opposed to oligonucleotide probes, the reporter (or terminalextension) of the new probe is a peptide that carries a series ofpositively-charged amino acids (“Peptide reporter” in FIG. 18A bracket)and the center domain is a peptide nucleic acid comprising nucleotidesthat are complementary to the target nucleic acid. When there are asufficient number of positively-charged amino acids in the reporter orterminal extension portion of the probe, the net charges of themiRNA/probe complex are still positive such that when the target miRNAbinds to the PNA (peptide nucleic acid) domain of the probe the entiremiRNA/probe complex forms a strong dipole molecule. We have engineered ananopore with a negatively-charged residue ring at the trans opening ofthe pore (S. aureus alpha-hemolysin comprising a K131D mutation). Thewild-type S. aureus alpha-hemolysin peptide sequence (National Centerfor Bioinformatics Accession NO. AAA26598.1); is:

(SEQ ID NO: 21) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

The variant S. aureus K131D alpha-hemolysin peptide sequence is:

(SEQ ID NO: 22) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTG D IGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

Therefore, the positively-charged peptide domain of the probe dipolewill be both pushed by the positive voltage (cis grounded) and attractedby the negative ring at the trans opening, guiding the trapping of themiRNA/complex into the n-barrel of the pore. At the positive voltage,any other free nucleic acids components will be repulsed from enteringthe pore due to the negative charge carried. This significantly reducessignals by free RNA components, and most observed events are either dueto the trapping of the miRNA/probe complex or the translocation of theprobe. The use of peptide-PNA probe enables selective detection of thetarget miRNA.

Results. FIG. 18A shows the diagram of the miRNA/probe complex. Thebracketed miRNA is target miRNA Let-7b. The bracketed probe P7b has abracketed “Peptide Reporter” part and a bracketed “PNA” (peptide nucleicacid) part. The PNA is for capturing Let-7b, and the bracketed “Peptidereporter” is a positively-charged peptide corresponding the sequence ofHIV-TAT, which contains +8e contributed by arginines and lysines. FIG.18B shows events for translocation of the peptide-PNA probe, P7b. Thecharacteristic events last for 3 ms and reduce the current to 10 pA at+180 mV. FIG. 18C shows no block events can be observed with free miRNAlet-7b (without probe) in the solution at +180 mV.

FIG. 18D shows signature events for the trapping of the let-7b/P7bcomplex. These events characteristically last for 100 ms and reduce thecurrent to 57 pA at +180 mV, completely different that for the probe.FIG. 18E shows that Let-7c, which has two different nucleotides fromLet-7b, cannot bind to PNA of the probe P7b, therefore does not generatesignature events as in FIG. 18C. Almost all observed events are due tothe probe itself.

FIG. 18F compares the duration-amplitude property for P7b binding toLet-7b (fully match, two separate clusters without overlay) and Let-7c(2 mismatches, two clusters fully overlay). This suggests an accuracy ofalmost 100% in differentiating sequence-similar miRNAs with twodifferent nucleotides.

Utilization of signature events to understand various molecularprocesses in the nanopore for biosensing applications is also provided.In FIG. 19A, we observed a novel type of three-level current patternwhen employing HP-C30 with a hairpin at the 3′-end of short strand. ItsLevel 1 and Level 2 are consistent with the unzipping of HP-C30 andtranslocation of the unzipped short strand from the nanocavity to theβ-barrel. However, the duration of Level 1′ was drastically prolonged by80 folds to 15±1.9 ms, compared to the target without a hairpin. Theprolonged Level 1′ is in agreement with the unzipping of hairpin priorto threading in the β-barrel. As many DNA or RNA structures such asaptamers contain hairpins, we can use this system and the signatureevents to study these structures and study their binding interactionwith their protein targets. In FIG. 19B, we also demonstrated a newmulti-level current pattern when using SA-C30 attached with astreptavidin at the 3′-end of the short strand. Again, both thefully-blocked Level 1 and partially-blocked level 2 are consistent withthe unzipping of SA-C30 and translocation of the unzipped short strandfrom the nanocavity to the β-barrel. The current stayed at Level 1′ forminutes until it was forced to recover by a negative voltage. The longterm Level 1′ can be interpreted by that although the short strand ofSA-C30 moves into the β-barrel after unzipping, its translocation isprevented by the attached large streptavidin. This result suggested thepotential of using signature events for protein detection. In FIG. 19C,we demonstrated that the complex can be sequentially unzipped in thenanopore in two steps when using a short oligonucleotide to link twoDNAs. The unzipping of the two DNAs can be clearly revealed by the twoLevel 2 states.

Conclusion. The peptide-PNA probe enables 1) selective detection of thetarget miRNA, 2) greatly enhanced accuracy in differentiatingsequence-similar miRNAs.

While the invention has been described in connection with specificembodiments thereof, it will be understood that the inventive device iscapable of further modifications. This patent application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure as come within known or customarypractice within the art to which the invention pertains and as may beapplied to the essential features herein before set forth and as followsin scope of the appended claims.

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Advanced Materials 18, 3149-3153 (2006).

What is claimed is: 1-13. (canceled)
 14. A method of detecting anoligonucleotide in a sample with a nanopore system, comprising the stepsof: a) mixing a sample suspected of comprising a target oligonucleotideand a probe, said probe comprising a center domain with a complementarysequence to said target oligonucleotide and a terminal extension taggedto at least one of its 3′ and 5′ terminals, to produce a sample mixturewherein said central domain of said probe can hybridize to said targetoligonucleotide, b) applying a voltage to said sample mixture in a ciscompartment of a dual chamber nanopore system sufficient to drivetranslocation of said hybridized probe and target oligonucleotidethrough a nanopore of said system by an unzipping process, and c)analyzing an electrical current pattern in said nanopore system overtime, wherein presence of said oligonucleotide in said sample isindicated by at least one signature electrical current block that isdistinct from a background electrical current block that occurs withsaid sample alone or said probe alone.
 15. The method of claim 14,wherein said signature electrical current block comprises at least oneof a: i) a current block of different duration than a background currentblock; ii) a different number of distinct current blockade levels than abackground current block_(;) iii) a different order of occurrence ofcurrent blockade levels than a background current block; iv) a differentcurrent amplitude at a blockade level than a background current block;v) a different current amplitude of each blockade level than abackground current block; or any combination of (i), (ii), (iii), (iv),or (v).
 16. The method of claim 14, wherein at least one terminalextension is a charged polypeptide or an oligonucleotide.
 17. The methodof claim 14, wherein said terminal extension comprises a chargedpolypeptide containing at least two positively charged amino acidsand/or at least two aromatic amino acid residues.
 18. The method ofclaim 17, wherein said charged polypeptide provides for a net positivecharge when said probe is hybridized to said target oligonucleotide. 19.The method of claim 14, wherein said nanopore comprises a negativelycharged residue ring at the trans opening of the pore.
 20. The method ofclaim 14, wherein said nanopore comprises an alpha-hemolysin variantcomprising a K131D, K131E, or K131H amino acid substitution.
 21. Themethod of claim 14, wherein steps (a) and (b) or step (b) are performedat a pH value of less than
 7. 22. The method of claim 14, wherein saidtarget oligonucleotide is a DNA molecule or an RNA molecule.
 23. Themethod of claim 22, wherein said RNA molecule is an miRNA or a fragmentthereof, and wherein said fragment comprises at least 15 nucleotides ofsaid RNA molecule.
 24. (canceled)
 25. The method of claim 14, wherein afrequency of said signature electrical current blocks is used toquantitate an amount of said target oligonucleotide in said sample. 26.The method of claim 14, wherein a non-target oligonucleotide is spikedinto said sample and a frequency of electrical current blocksattributable to said non-target oligonucleotide is used to normalizesaid frequency of signature electrical current blocks.
 27. The method ofclaim 14, wherein said center domain of said probe comprises at leastabout 15 nucleotides or nucleobases that are complementary to the targetoligonucleotide.
 28. A method for detecting two or more distinct targetoligonucleotides that differ by at least one nucleotide in a sample witha nanopore system, comprising the steps of: a) mixing a sample suspectedof comprising said distinct target oligonucleotides and a probe, saidprobe comprising a center domain with a fully or partially complementarysequence to said target oligonucleotides and a terminal extension taggedto at least one of its 3′ and 5′ terminals, to produce a sample mixturewherein said central domain of said probe can hybridize to said targetoligonucleotides, b) applying a voltage to said sample mixture in a ciscompartment of a dual chamber nanopore system sufficient to drivetranslocation of hybridized probe and target oligonucleotides through ananopore of said system by an unzipping process, and c) analyzing anelectrical current pattern in said nanopore system over time, whereinpresence of two or more distinct target oligonucleotides in said sampleis indicated by two or more distinct signature electrical currentblocks.
 29. The method of claim 28, wherein said two or more distinctelectrical current blocks comprise at least one of: i) differentsignature electrical current block durations (τsig); ii) a differentnumber of distinct current blockade levels; iii) a different order ofoccurrence of current blockade levels; iv) a different currentamplitude; v) a different current amplitude of each blockade level; orany combination of (i), (ii), (iii), (iv), or (v).
 30. (canceled) 31.The method of claim 28, wherein said target oligonucleotides are DNAmolecules or RNA molecules.
 32. The method of claim 31, wherein said RNAmolecules are miRNAs or fragments thereof, and wherein said fragmentscomprise at least 15 nucleotides of said RNA molecule. 33-34. (canceled)35. The method of claim 28, wherein the terminal extension is a chargedpolypeptide or an oligonucleotide.
 36. The method of claim 35 28,wherein the charged polypeptide comprises at least two positivelycharged amino acids and/or at least two aromatic amino acid residues.37. The method of claim 36, wherein said charged polypeptide providesfor a net positive charge when said probe is hybridized to said targetoligonucleotide.
 38. The method of claim 36, wherein said nanoporecomprises a negatively charged residue ring at the trans opening of thepore.
 39. The method of claim 36, wherein said nanopore comprises analpha-hemolysin variant comprising a K131D, a K131E, or a K131H aminoacid substitution. 40-44. (canceled)
 45. The method of claim 28, whereinsaid central domain comprises at least about 15 nucleotides ornucleobases that are complementary to the target oligonucleotide. 46-49.(canceled)
 50. A kit comprising: (a) a probe comprising a center domainwith a fully or partially complementary sequence to a targetoligonucleotide and a terminal extension tagged to at least one of its3′ and 5′ terminals, wherein said central domain of said probe canhybridize to the target oligonucleotide; and, (b) a nanopore.
 51. Thekit of claim 50, wherein said probe comprises at least one terminalextension that is a charged polypeptide.
 52. The kit of claim 50,wherein the charged polypeptide comprises at least two positivelycharged amino acids and/or at least two aromatic amino acid residues.53. The kit of claim 52, wherein said charged polypeptide provides for anet positive charge when said probe is hybridized to a targetoligonucleotide.
 54. The kit of claim 50, wherein said nanoporecomprises a negatively charged residue ring at the trans opening of thepore.
 55. The kit of claim 50, wherein said nanopore comprises analpha-hemolysin variant comprising a K131D, a K131E, or a K131H aminoacid substitution. 56-60. (canceled)